Implantable xenografts and prosthetic devices are typically sterilized prior to implantation in an intended recipient. Sterilization is required to ensure that the devices do not introduce potential pathogens, or other biologically detrimental agents into the intended recipient. Sterilization is particularly relevant where biomaterials from human or other mammalian donors are constituents of the graft or device. For example, U.S. Pat. No. 6,214,054 to Cunnanen et. al (incorporated herein in its entirety) includes discussion of well recognized sterilization techniques for such biological tissues. Device components are sterilized individually prior to assembling the device or, alternatively, they are sterilized by the process of “terminal sterilization”. In the terminal sterilization process, the device is sterilized following its construction, i.e., after all the components have been combined with one another in the device. Both processes may be used in combination to ensure complete sterilization of the graft or device. A variety of physical or chemical methods have been developed for use in sterilization and include, for example, exposure to chemicals or heat, or exposure to ionizing or non-ionizing radiation. These methods, however, have inherent problems. Moreover, most of the methods are inappropriate for bioprosthetic devices incorporating mammalian tissue.
Exemplary sterilization methods include treating prosthesis and graft components with chemical reagents. The chemical reagents themselves, or reaction byproducts derived from the reagents, can be harmful to the intended recipient of the prosthetic device. Accordingly, such chemicals must be removed prior to implantation of the devices. Common chemical sterilizing agents include ethylene oxide and formaldehyde, both of which are alkylating agents and, therefore, can modify and inactivate biologically active molecules. Both of these chemicals are, however, known to be carcinogens and mutagens (Davis et al., (1973) “Microbiology, 2nd Ed.”, Harper and Row, Publishers).
Other methods of sterilizing device components include exposing the device or components thereof to plasma (Moulton et al., U.S. Pat. No. 5,084,239) heat, or ionizing radiation. Similar to chemical treatment, however, where the device includes biological components (e.g. proteins, cells, tissues), exposing the device to elevated temperatures, radiation or plasma is not desirable because proteins and other biological materials can be denatured and subsequently inactivated or weakened by exposure to these forms of energy. Although the sterilization of objects by exposure to ionizing and non-ionizing radiation obviates the necessity of adding potentially toxic chemicals, the radiation energy and/or its byproducts, including oxygen free radicals, are competent to modify protein conformation and so can damage or destroy proteins, cells and tissue. In addition, exposure of some medically important polymers, for example, polyurethane or polymethylmethacrylate to gamma radiation can result in immediate and long term physical changes to the polymer. Moreover, irradiation with gamma or beta rays does not destroy all pathogens with certainty. Indeed, certain viruses are radiation resistant. Thus, alternatives to sterilizing xenografts and biologically derived components of prosthetic devices with chemicals and radiation are being avidly sought.
The chemical- and radiation-based methods for sterilizing biologically derived prosthetic device components described above, rely on the inactivation of infectious agents associated with the biological component of the device. The inactivated infectious agent generally remains associated with the biological component of the device. A promising mode of rendering a biologically derived material non-infectious relies on inactivating infectious agents by contacting them with supercritical fluids. Little attention, however, has been focused on the use of supercritical fluids to extract infectious materials from mammalian soft-tissue.
Supercritical fluids have principally been used in the field of chromatography, where these dense materials are employed as extraction solvents and interactive mobile phases. For example, U.S. Pat. Nos. 4,816,159 and 4,880,543 to Khosah et al., each disclosing supercritical fluid chromatography methods that utilize specific packing materials. The packing materials disclosed in these patents are selected from metal oxide/hydroxide support materials having phosphorous-containing organic molecules bonded to reactive sites on the support materials. Historically, supercritical fluid chromatography has its origins in the mid-1960s, while its extraction analogue has only recently seen application in the field of analytical chemistry.
Extraction methods utilizing supercritical fluids have also been utilized for a number of applications. Distinct from chromatography using supercritical fluids, supercritical fluid extraction is a technique whereby organic compounds can be extracted from sample matrices utilizing a dense gas-like material, such as supercritical carbon dioxide. The solvation power of carbon dioxide is increased as the pressure and temperature are increased above their critical points, which are 1070 psi and 31° C., respectively. For example, U.S. Pat. No. 4,547,292 to Zarchy; U.S. Pat. No. 4,770,780 to Moses; U.S. Pat. No. 4,824,570 to Bethuel et al; U.S. Pat. No. 4,877,530 to Moses; and International Patent No. WO 85/04816 each disclose various processes or systems useful for practicing extraction utilizing supercritical fluids. Other references discussing supercritical fluid extraction include, Favati et al., J. Food Science 53: 1532–1536 (1988); King J. Chromatographic Science 27: 355–364 (1989); and King et al. J. Agricultural & Food Chemistry 37: 951–954 (1989).
A number of references disclose the use of supercritical fluids to inactivate microbes and viruses. For example, the mechanical destruction of microbial cells by sudden decompression in carbon dioxide CO2 in the supercritical state has been described (Nakamura et al., Biosci. Biotech. Biochem. 58(7): 1297–1301 (1994)). Castor et al. (U.S. Pat. No. 5,877,005) disclose a method for inactivating a virion in a fluid containing a biological material. Bone grafts have been prepared by extracting lipid materials from the solid framework of the graft using supercritical fluid extraction. For example, EP-A-0.603.920 describes a process for the treatment of bone tissues in which a fluid in the supercritical state is used to extract lipidic organic matter. Supercritical fluid extraction has been used to remove both lipidic material and viruses from bone material. Representative references disclosing the removal of viruses from bone include, Fages et al., 21st Annual Meeting of the Society for Biomaterials, San Francisco, Calif., p. 238 (1995); Fages et al., Biomaterials 15: 650–656 (1994); Fages, U.S. Pat. Nos. 5,723,012; and 5,725,579.
The references set forth above disclose processes for inactivating infectious agents in bone and cartilage using supercritical fluids. The references disclose that to obtain implantable tissue, the bone graft material must be subjected to a number of steps in addition to the supercritical fluid extraction, including chemical treatment (with hydrogen peroxide) and/or enzyme treatment (protease) for extracting residual proteins. The tissue is then washed, dehydrated and disinfected in baths of ethanol (this last stage increasing safety as regards infection of the biomaterial). None of the references suggests that infectious materials can be effectively removed from soft-tissue by supercritical fluid extraction. Moreover, none of the references suggests that treatment of any tissue with supercritical fluids alone will remove infectious agents from the tissue.
Of increasing concern is the presence of infectious prions in biologically derived materials used for xeonografts and prosthetic devices. The widespread occurrence of prion-related disease and the possibility of interspecies transmission has serious implications for the biotechnology industry, which derives many of its products from mammalian tissue (Di Martino Biologicals 21 : 61–66 (1993)). Concerns about the safety of mammalian tissue products has led to studies on the inactivation of prions. These studies indicate that prions are more resistant toward inactivation than more conventional pathogens such as viruses or bacteria. Thus, relatively harsh conditions are required to decontaminate prion-containing biological materials. The only methods currently known to disinfect prion contaminated biological preparations are prolonged autoclaving at 130° C. or above, and treatment with concentrated sodium hydroxide solution. These methods have been recommended for routine inactivation of prions (Department of Health and Social Security Circular 84: 16 (1989)). It has also been reported that 100 kD cutoff ultrafiltration in combination with treatment with 6M urea results in decontamination of prion containing preparations (Pocchiari et al., Arch. Virol. 98: 131–135 (1988)). Other methods capable of lowering prion activity include treatment with organic solvents, detergents, protein-denaturing agents, chaotropic salts and phenol (Millson et al., in Prusiner and Hadlow, eds. SLOW TRANSMISSIBLE DISEASES OF THE NERVOUS SYSTEM, vol. II. New York: Academic Press 409–424 (1979); Prusiner et al., PNAS 78: 4606–4610 (1981); Kimberlin et al., J. Neurol. Sci. 59: 390–392 (1983); Walker et al., Am. J. Public Health 73: 661–665 (1983); Brown et al., J. Infect. Dis. 153: 1145–1148 (1986)).
The extreme conditions required to eliminate infectivity, and particularly prion infectivity, in biomaterials are typically incompatible with methods intended to preserve the useful activity and structure of these materials. The harsh conditions of prior methods are particularly deleterious to mammalian soft-tissue, resulting in the denaturation of functional and structural components of the tissues. There is, thus, a need for a method for removing infectious materials from mammalian soft-tissues that does not compromise the integrity of these desirable biomaterials.